Aging can be generally characterized as the long-term loss of tissue architecture, function and regenerative capacity. In the previous funding period, we explored the effects of two key challenges to long-term tissue maintenance using a novel system to delete the ATR checkpoint kinase in adult mice. We showed 1) that exhaustion of regenerative potential through stem cell attrition and increased replicative demand accelerates the appearance of age-related pathologies, and 2) that failure to suppress the accumulation of highly-damaged cells can dominantly inhibit tissue regeneration. This later mechanism putatively serves as a tissue renewal checkpoint that prevents regeneration until damaged cells can be effectively cleared. Finally, our preliminary results indicate that delayed renewal is immediately followed by a highly stimulatory phase that ultimately accelerates degeneration. Herein, we propose to further develop these research areas by defining the physiological conditions that promote replication-associated DNA damage and correlating this damage with debilitated stem cell potential. To accomplish this goal, hypomorphic ATR suppression will be used to convert transient replication abnormalities into more long-lived intermediates (double strand breaks). This system will permit the identification of both cell populations and genomic loci that are selectively susceptible to replication abnormalities during compensatory renewal. In addition, we propose to use our ATR-conditional system to characterize how DNA-damaged cells coordinate the distinct phases of regeneration through extrinsic factors. These factors include ones that that inhibit renewal and those that subsequently stimulates it. In aggregate, these studies will determine how urgent episodes of compensatory renewal are regulated and how these events can lead to the decline of long-term renewal potential. PUBLIC HEALTH RELEVANCE: Age-associated pathologies are a major contributor to morbidity and mortality in the United States. Occurrence of these diseases is strongly influenced by the effectiveness of organ maintenance and tissue regeneration. In this proposal, we describe experiments to both better define the causes of debilitated regenerative potential with age and investigate the mechanisms used to renew tissues following acute loss of tissue integrity.